Effects of spaceflight on the EEG alpha power and functional connectivity

Electroencephalography (EEG) can detect changes in cerebral activity during spaceflight. This study evaluates the effect of spaceflight on brain networks through analysis of the Default Mode Network (DMN)'s alpha frequency band power and functional connectivity (FC), and the persistence of these changes. Five astronauts' resting state EEGs under three conditions were analyzed (pre-flight, in-flight, and post-flight). DMN’s alpha band power and FC were computed using eLORETA and phase-locking value. Eyes-opened (EO) and eyes-closed (EC) conditions were differentiated. We found a DMN alpha band power reduction during in-flight (EC: p < 0.001; EO: p < 0.05) and post-flight (EC: p < 0.001; EO: p < 0.01) when compared to pre-flight condition. FC strength decreased during in-flight (EC: p < 0.01; EO: p < 0.01) and post-flight (EC: ns; EO: p < 0.01) compared to pre-flight condition. The DMN alpha band power and FC strength reduction persisted until 20 days after landing. Spaceflight caused electrocerebral alterations that persisted after return to earth. Periodic assessment by EEG-derived DMN analysis has the potential to become a neurophysiologic marker of cerebral functional integrity during exploration missions to space.

www.nature.com/scientificreports/ Due to the complexity of cognitive function, neurobehavioral assessments of astronauts, cosmonauts, and taikonauts have relied primarily on neuropsychological tests 3,5,10,21,22 . These neuropsychological tests are immensely useful in evaluating the subject's performance, but their capabilities in assessing preclinical and subclinical changes can be limited 23 . Structural changes have been detected in brain MRIs of astronauts a few days after having returned from a 6-month mission aboard the International Space Station (ISS). It has been estimated that greater than 50% of the personnel may be affected by such structural changes 18,19,21 . We view that the development of auxiliary assessment and monitoring methods and neuropsychological tests is critical. However, mission weight limit and microgravity environment are technical constraints. In this context, electroencephalography (EEG) has proven to be a technically feasible procedure in both transport and operational aspects 24,25 .
EEG, a reliable neurophysiological technique that measures neuronal electrical activity, has been used in spaceflight as a part of polysomnography 26 . An in-flight microgravity environment has been reported to affect EEG findings by Cheron et al. in the form of a transient alpha power increase during the arrest reaction 27 . Although the significance of this finding is not well understood, the alpha band is known to be a crucial frequency domain of oscillatory brain activity, and has been associated with attention, inhibition, and working memory 28,29 .
In this context, the EEG-derived Default Mode Network (DMN) is of particular interest 41 . As laid out by Cheron et al. the DMN is a global workspace that cannot be regarded as "a simple resting state" but as a complex process involving dynamic interplay between conscious and unconscious states 42 . Moreover, this network includes several cortical regions (e.g., posterior cingulate cortex, precuneus, medial prefrontal, and inferior parietal cortices) 43 related to different neurocognitive disorders [44][45][46] . Furthermore, EEG measurements of these networks are readily feasible from an operational relevance point of view. The importance of functional assessment using EEG is further supported by the fact that alterations in brain function can precede structural brain abnormalities 47 . The validity of this method has been proven in clinical subjects 32 . We view that the brain's functional connectivity evaluation has excellent potential to be used as a practical assessment and monitoring tool during spaceflight.
The analyzed dataset used in this study is a part of the NEUROSPAT experiment 6,25 . We performed a retrospective analysis of the EEG data, obtained during long-duration ISS missions, to evaluate functional brain networks under three different conditions: pre-fight ground level, in-flight extraterrestrial, and post-flight ground level. Our objective is to assess the alterations of DMN's alpha frequency band power and FC, and the persistence of these changes. We hypothesize that EEG-derived DMN would be disrupted by the flight condition, and FC between brain nodes would be reduced.

Methods
The dataset originated from NEUROSPAT experiment (AO-2004, 118) 6,8,16 . Five male astronauts (54.2 ± 2.6 years old) with 6 months in low earth orbit (174.6 ± 19.9 days) participated in this experiment. The Ethics Committee of the Faculty of Medicine of the Université Libre de Bruxelles, the European Space Agency Medical Care Committee, and the NASA Johnson Space Center Institutional Review Board for Human Testing approved all experimental protocols and procedures, which were performed following the Helsinki Declaration of 1964. To ensure comparable levels of sleep quantity the night before the recordings, a sleep questionnaire was filled out by astronauts. Astronauts were allocated 8.5 h of sleep the night before the experiment. The experiments were not performed in the 48 h following air travel that involved a change of > 4 time zones, nor following work shifts inducing > 4 h of time shift, nor the day after imposed sleep deprivation, nor after a highly strenuous mental or physical task such as centrifuge training, vestibular counter-measures experiments, and extravehicular activities.
Astronauts were asked to maintain their normal consumption of caffeine and were not allowed to take alcohol or medication 16 h before the experiment. Each astronaut was asked to execute the experiments on approximately the same day time preferably in the morning ± 2 h. Participants were assessed in three conditions: (1) pre-flight (on earth; three times: 66.8 ± 9.0, 42.6 ± 0.9, and 28.0 ± 0.4 days before departure), (2) in-flight (aboard ISS station in space; two times: 8.8 ± 1.8 and 54.6 ± 3.7 days during mission) and 3) post-flight (on earth; four times: 3.0 ± 0.4, 7.0 ± 1.2, 16.8 ± 0.64 and 20.2 ± 1.04 days after arrival).

Ethics statement. The European Space Agency Medical Care Committee and the NASA Johnson Space
Centre Institutional Review Board for Human Testing approved all experimental procedures, which were performed in accordance with the Helsinki Declaration. All participants gave written, informed consent prior to starting the experiment.
EEG data acquisition. The brain activity of all participants was measured by electroencephalography (EEG) during 2-min resting-state eyes-closed (EC) and 2-min resting-state eyes-open (EO) conditions 6  www.nature.com/scientificreports/ participants were seated comfortably in a chair. During microgravity in space, they stayed in a free-floating condition where significant trajectory shifts were prevented using a belt around the subject's waist, which was attached to straps fixed to metal rings located at the racks on both sides of the Columbus module of the ISS. To avoid any external visual distractors, a cylindrical tube attached to the laptop screen including a face mask was fitted to the astronaut's head. This recording setup was used both for the ground and ISS measurements. EEG data were recorded at a sampling frequency of 1116 Hz using the 59-channel electroencephalogram mapping module (MEEMM) of the European physiology module, installed in the Columbus module of the ISS, at the European Astronaut Center (Köln, Germany) or in Star City (Moscow). The MEEMM uses a dedicated physical reference electrode (right earlobe). For some post-flight recordings at the Johnson Space Center (Houston), an asalab 64-channel amplifier (ANT Neuro BV, Hengelo, Netherlands) was used at ground level in a standard lab environment. The asalab amplifier is a stationary DC-EEG amplifier with a common average reference. Scalp electrode impedance was measured and kept below 5 kΩ for all recordings.
EEG data analysis. Source activity was estimated using a template (Montreal Neurological Institute-MNI-space) with a regular volumetric grid of 10 mm spacing. The template was linearly transformed to fit the head shape of each participant (individual subjects' T1 MRI were not available). Sources were reconstructed independently for each subject through the exact low-resolution brain electromagnetic tomography (eLORETA) method 50 using a regularization factor of 10 −8 . Each source position was labeled using the Automated Anatomical Labeling (AAL) atlas 51 . Only those sources labeled as part of one of the 22 cortical areas involved in the default mode network (DMN) were included in subsequent analyses (215 sources).
The power spectrum of each source was computed for each epoch using the fast Fourier transform with Hanning tapers with 0.25 Hz smoothing. Relative power was calculated by normalizing each ROI's spectrum by the total power over the 2-45 Hz range. The average power of the DMN per each classical frequency band was calculated by averaging all epochs, all sources, and all corresponding frequency steps, resulting in a sourcereconstructed power matrix of 9 stages of 5 frequency bands in 5 participants.
FCs between the 22 DMN ROIs (representing the 22 cortical areas involved in the DMN) were assessed with the corrected imaginary phase-locking value (ciPLV) 52 , a phase synchronization measure that evaluates the distribution of phase differences extracted from two ROIs time series and is insensitive to zero-lag effects, as it removes the contribution of the zero phase differences 52 . Symmetrical, whole-brain matrices of 215 × 215 sources were thus obtained by averaging ciPLV values across epochs for each participant and frequency band. Then, DMN 22 × 22 ROIs FC matrices were constructed by averaging the FC weights of the corresponding sources for each pair of ROIs. Lastly, we computed the strength of each ROI (also known as weighted global connectivity), defined as the average FC across all links that belong to the DMN network. Then each ROI's strength was normalized by dividing the number of links connected to it, obtaining one brain map of normalized ROI strengths per participant and frequency band to account for the number of links.
Statistical analysis. Statistical analysis was performed using Prism 9 software (GraphPad version 9.0.0 https:// www. graph pad. com/, San Diego, CA, USA). The final values of power spectrum and FC obtained under the three conditions pre-flight, in-flight, and post-flight were statistically compared. Eyes-closed and eyesopened sub conditions were compared to determine differences in the DMN alpha band relative power and FC strength. Depending on the number of independent variables, normally distributed data (Shapiro-Wilk normality test), and equality of variances (sphericity test) of the groups compared, we used one-way ANOVA or two-way Repeated Measure ANOVAs by Tukey's multiple comparison test. Geisser and Greenhouse's correction method was used for repeated measures with reduced sphericity. F values, t values, and degrees of freedom are reported in Table S1 of the supplementary material. Results are reported as mean ± standard error (SEM) and p values coded as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Results
Alpha band power changes in the DMN. All individual subjects showed a reduction of DMN alpha band power during eyes-closed (EC) under in-flight conditions (aboard ISS station) (Fig. 1). As a cohort, the DMN alpha band power (EC) was found to be significantly decreased (F = 24.09, p < 0.001, eta2 = 0.53) during the in-flight condition when compared to the pre-flight (p < 0.001) and post-flight conditions (p < 0.01) (Fig. 1a). These changes were observed across different DMN regions (Fig. 1b-d) www.nature.com/scientificreports/ observed during the in-flight condition compared to the pre-flight condition (Fig. 1b). Precuneus, the anterior cingulate cortex, and parietal regions showed the most considerable differences in alpha band power (higher q value). For a detailed description of the statistical analysis of the ANOVA, effect size of all the DMN regions refer to Table S1 of the supplementary material. Additionally, we evaluated the DMN alpha band power during eyes-open (EO) and found a reduction of alpha power (F = 12.37, p < 0.001, eta2 = 0.39) across all subjects under in-flight conditions compared to pre-flight conditions (Fig. 2). As a cohort, the DMN alpha band power (EO) was found to be significantly decreased during the in-flight condition when compared to pre-flight (p < 0.05) (Fig. 2a).
Notably, DMN alpha band power (EC/EO) continued to be reduced in all subjects during post-flight conditions compared to pre-flight (Figs. 1a, 2a). As a cohort, the reduction of DMN alpha band power during the postflight condition was statistically significant when compared to in-flight condition (EC: p < 0.001; EO: p < 0.01). These changes were observed across different DMN regions (Fig. 2b-d).
There were DMN alpha band power differences between eye-closed and eyes-opened conditions. Higher relative power was found during eye-closed in all flight conditions, which were statistically significant as a cohort (pre-flight: p = 0.0008; in-flight: p < 0.0001; post-flight: p = 0.0033) (Fig. 3a-c).  (Fig. 4a). These changes were observed across different DMN regions (Fig. 4b-d). Similar to alpha band power alterations, significant changes in alpha band FC strength were mainly observed during in-flight conditions compared to the pre-flight condition (Fig. 4b), with the parietal regions showing the most significant differences (highest q value). For a detailed description of the statistical analysis refer to Table S1 of the supplementary material.

Changes in alpha band
Additionally, we evaluated the DMN alpha band FC strength during eyes-opened (EO) and found a reduction of FC strength (F = 17.21, p < 0.001, eta2 = 0.39) in all subjects under in-flight conditions compared to pre-flight conditions (Fig. 5). As a cohort, the DMN alpha band FC strength (EO) was found to be significantly decreased  www.nature.com/scientificreports/ during the in-flight condition when compared to pre-flight (p < 0.01) (Fig. 5a). Moreover, post-flight conditions (EO) also showed a significant decrease in FC strength compared to pre-flight (p < 0.01). These patterns were noticed across different DMN regions (Fig. 5b-d).
There were DMN alpha band FC strength differences between eye-closed and eyes-opened conditions. Higher FC strength was found during eye-closed in all flight conditions (Fig. 6a-c). However, these differences were only statistically significant as a cohort at ground level (pre-flight: p = 0.0059; post-flight: p = 0.0002), but not during in-flight aboard ISS (p = 0.1245).

Discussion
The impact of spaceflight and microgravity on cognitive function has been addressed by researchers and medical experts in space agencies, as optimal performance can be critical to mission success 9 . Thus, there are several studies 14 addressing neurophysiological alterations due to spaceflight, they include structural changes of grey matter and even in cerebrospinal fluid (CSF) 4 . Additionally, changes in visuo-attentional activity, visuospatial performance, brain activity, and effects of space travel on sleep quantity have been observed 4 . Our study findings indicate that spaceflight can produce neurophysiological alterations, as evidenced through EEG-derived DMN analysis. These changes result in a reduction of both DMN alpha band power and FC strength, notably over the frontoparietal regions, which occurred in space and persisted after return to earth across all five subjects.
We consider that these data can be of critical importance and warrant further investigation. The frontoparietal network has been implicated in task initiation and error control 53 . Changes to this network that we have observed for the in-flight condition may be of critical importance due to the direct impact on task performance aboard the spacecraft. Moreover, alpha band reduction in power or connectivity has been reported in various neurocognitive disorders such as dementia, stroke, and traumatic brain injury 32,54 . The persistent changes after return to earth in our cohort are notable, indicating that effects of spaceflight are present at least 20 days after return to earth.
The changes in our cohort cannot be readily explained by their expected high task demand and stressful environment. In contrast to the alpha power reduction observed in our astronaut cohort, commercial pilots whose jobs also involve multitasking and situations of high cognitive stress level were reported to have increased  www.nature.com/scientificreports/ alpha band power 55 . Common occupational cognitive-related conditions such as high working memory load 56 , drowsiness during driving 57 , long periods of psychoacoustic stimulation 58 , and mental exhaustion 59 are also associated with an increase in alpha power. The unique spaceflight conditions are a probable cause of DMN alpha band power and FC strength reduction. Simulated microgravity environments such as head-down or bed rest 60 have been associated with changes in alpha power (i.e., reduce alpha power) 61 . However, these alterations are transient and returned to baseline after a return to normal positioning, and there were no functional connectivity differences 61 . EEG has also been used in space to measure changes in neurocognitive performance and brain activity that result from exposure to microgravity and isolation 62 . Alterations in brain activity are commonly identified in the faster EEG frequencies, demonstrating inhibition of brain activity in the cortical regions under these conditions 4 . Additionally, long-term isolation and dry immersion studies have found alterations in sleep spectral content 63 and changes in alpha power as well 64 . Nonetheless, these effects are expected to disappear after weeks of returning to normal social activity. In our study, we demonstrate both persistent reductions of alpha band power on DMN upon return to earth and frontoparietal functional connectivity changes, indicating that microgravity alone would not explain these findings. Our results showed reduced brain activity that persisted weeks after landing and resumption of social activity, making it unlikely that isolation is the sole contributing factor to the alpha band reduction.
Galactic cosmic radiation (GCR) 65 is an important risk factor for human spaceflight, especially for expeditionary missions beyond lower earth orbit (LEO). Radiation-induced brain injury is a known condition and has been linked to cognitive impairment 66 . Gamma radiation exposure has been reported to trigger oxidative stress, increase inflammation, and cause neurotransmitter fluctuations in the central nervous system in rats, resulting in the presence of β-amyloid deposits in the cerebral cortex 67 . β-amyloid deposits have been notably associated with Alzheimer's disease 68 , and EEG findings indicate reduced alpha band power and abnormal frontoparietal coupling 69,70 . Although our data were recorded in lower earth orbit, the observed reduction of the alpha power and connectivity in our study could be in part caused by cascading patho-mechanisms induced by this proteinopathy, as it happens in Alzheimer's disease 71 .
These EEG results are similar to our cohort's observations, raising questions regarding the influence of radiation on our subjects and findings. Even under the protection of the earth's magnetic field, astronauts aboard ISS receive a 40-60 times higher effective yearly dose-rate than on earth due to galactic cosmic radiation, solar flares, and radiation from the Van Allen Belt 72 .
There are many remaining questions that the available data of this study alone cannot be fully addressed. The onset and progression of DMN changes during spaceflight cannot be reliably deduced from only two recordings, averaging 9 and 55 days, which were available for our analysis. The duration and return-to-baseline of these changes are also unclear, as follow-up recording continued only until 20-days after return to ground level. The number of participants and gender balance in this study is also a limiting factor. Due to the nature of our dataset, it was not possible to include both sexes. In 62 years of human spaceflight activities, only 6 female astronauts and cosmonauts were involved in inflight EEG studies compared to more than 70 males 73,74 . Our dataset includes only EEGs from male astronauts. Moreover, it is known in the literature that there are gender differences in neurophysiological data related to the alpha band in 1G conditions 75,76 and even in hypo-and hyper-gravity conditions 77 . We expect to find differences between gender in future studies. These issues should be resolved as exploration missions beyond LEO and increased commercial spaceflights activities are at the near horizon. We notice that future studies are needed to determine the onset and persistence of these changes in a larger cohort. Traditionally used neuropsychological testing alone has limits, as neuropathology suggests cognitive impairment can develop prior to changes in performance 23 . Future studies of EEG-derived DMN analyses, in conjunction with neuropsychological testing and imaging, can promote understanding of the spaceflight effect on neurobiology, ensuring mission success and crew safety. Furthermore, EEG-derived DMN analysis carries excellent potential as a neurophysiologic marker with practical applications, as it can be readily performed and analyzed during spaceflight missions 78 .

Data availability
The data that support the findings of this study are available upon reasonable request to the corresponding author.